40 Essays in Biochemistry 



indeed, an excretion, or merely the oozings from dying cells and, finally, 

 whether the phenomenon is an attribute of starving cells only. 



Since the technique of the experiments bears directly on the answers 

 to the above questions, the salient experimental procedures must be 

 given. For the starvation experiments bacteria were raised on a syn- 

 thetic medium to logarithmic growth phase from small inocula. They 

 were harvested by centrifugation when they reached a cell count of 

 not higher than 2 X 10 8 cells per milliliter and were resuspended after 

 sterile washing in a fresh medium lacking the nutrient of which they 

 were to be starved. When cellular concentrations higher than 2 X 10 8 

 cells per milliliter were desired, the bacteria were resuspended to an 

 appropriately smaller final volume. This precaution is essential to 

 insure that the experiments are performed with a bacterial population 

 which is preponderantly viable and which approaches physiological 

 homogeneity. For the study of the kinetics of the excretion, aliquots 

 of these aerobically incubating, starving, bacterial suspensions were 

 taken at intervals; the bacteria were eliminated by high-speed cen- 

 trifugation, and the absorption at 260 m^ of the supernatant fluid was 

 measured. At the same time, aliquots of the bacterial suspension were 

 diluted appropriately for plating for viable cells. No change in the 

 number of viable cells could be detected during the first 6 hours of 

 starvation. 



In Fig. 2 a study of the kinetics of the excretion of ultraviolet - 

 absorbing substances by E. coli K 12 W-6 on methionine and on glucose 

 starvation is represented. Analysis of the excreted products indicated 

 the presence of free bases and of nucleotides, but the relative quantities 

 were different on the two types of starvation. 



There are several lines of evidence to indicate that the nucleic acid 

 fragments which accumulate in the medium during the first 5 to 6 hours 

 of starvation are excreted products rather than the accumulated debris 

 from dying bacteria. The accumulation is considerably larger on 

 methionine starvation than on glucose starvation, yet, on prolonged 

 incubation, the bacteria remain fully viable for a longer time on 

 methionine starvation. Moreover, comparison of the output of ultra- 

 violet-absorbing material in a culture of 10° cells per milliliter with 

 that in a culture of 10 8 cells per milliliter revealed that there was a 

 greater output per cell at the lower concentration, but the measurable 

 death rate, on prolonged incubation, was greater at the higher cell 

 concentration. Finally, the kinetics of the excretion point either to 

 an exhaustion of excretable products or to some equilibrium, for a 



